Abstract Juxtacrine signaling mediates cell-cell communications via direct molecular interactions at the signaling interface, during development, synapse formation and remodeling, immune activities, and tissue formation. Despite increasing knowledge of these signaling events, little is known about how the juxtacrine receptors sense and regulate cell signaling in response to the dynamic changes of its surrounding cells. The challenge of interrogating spatiotemporal dynamics of juxtacrine cell-cell signaling stems from the fact that many juxtacrine receptors integrate chemical, spatial, and mechanical cues to differentially regulate cell signaling. To deconstruct and decode the working mechanisms of these receptors with high spatiotemporal complexity, new technology tools allowing manipulation of the individual cues with different modes of stimulation, while reporting cellular responses with high spatiotemporal precision. Toward this aim, we previously developed nanotechnology platforms including monovalent quantum dot (mQD) probes, mechanogenetics, nanoruler force microscopy (NRFM), and magnetically amplified protein-protein interaction (MAP-I) tools. mQDs report single molecule trajectories of the targeted receptors, providing its dynamic spatial and diffusion properties precisely. Mechanogenetics allows us to manipulate chemical, spatial, and mechanical properties of the targeted receptors, while monitoring cellular responses to the respective cues. NRFM enables us to investigate force-responsive structural changes of the target receptors, and hence provides important insights into the mechanism of mechanotransduction. MAP-I allows for ultrasensitive detection of protein-protein interactions through magnetic amplification, enabling identification of weak protein-protein interactions that have not been possible with any other technologies. By using these new technologies, here, we propose to investigate the interaction and signaling dynamics of Notch and Neuroligin, key signaling proteins in development and synaptic function, respectively. Ultimately, we aim to provide a platform technology for the systematic investigation of operating principles for a wide range of juxtacrine signaling, accelerating our understanding of cell-cell communication.